Anti-tumor activity of all-trans retinoic acid in gastric-cancer: gene-networks and molecular mechanisms

Background Gastric-cancer is a heterogeneous type of neoplastic disease and it lacks appropriate therapeutic options. There is an urgent need for the development of innovative pharmacological strategies, particularly in consideration of the potential stratified/personalized treatment of this tumor. All-Trans Retinoic-acid (ATRA) is one of the active metabolites of vitamin-A. This natural compound is the first example of clinically approved cyto-differentiating agent, being used in the treatment of acute promyelocytic leukemia. ATRA may have significant therapeutic potential also in the context of solid tumors, including gastric-cancer. The present study provides pre-clinical evidence supporting the use of ATRA in the treatment of gastric-cancer using high-throughput approaches. Methods We evaluated the anti-proliferative action of ATRA in 27 gastric-cancer cell-lines and tissue-slice cultures from 13 gastric-cancer patients. We performed RNA-sequencing studies in 13 cell-lines exposed to ATRA. We used these and the gastric-cancer RNA-sequencing data of the TCGA/CCLE datasets to conduct multiple computational analyses. Results Profiling of our large panel of gastric-cancer cell-lines for their quantitative response to the anti-proliferative effects of ATRA indicate that approximately half of the cell-lines are characterized by sensitivity to the retinoid. The constitutive transcriptomic profiles of these cell-lines permitted the construction of a model consisting of 42 genes, whose expression correlates with ATRA-sensitivity. The model predicts that 45% of the TCGA gastric-cancers are sensitive to ATRA. RNA-sequencing studies performed in retinoid-treated gastric-cancer cell-lines provide insights into the gene-networks underlying ATRA anti-tumor activity. In addition, our data demonstrate that ATRA exerts significant immune-modulatory effects, which seem to be largely controlled by IRF1 up-regulation. Finally, we provide evidence of a feed-back loop between IRF1 and DHRS3, another gene which is up-regulated by ATRA. Conclusions ATRA is endowed with significant therapeutic potential in the stratified/personalized treatment gastric-cancer. Our data represent the fundaments for the design of clinical trials focusing on the use of ATRA in the personalized treatment of this heterogeneous tumor. Our gene-expression model will permit the development of a predictive tool for the selection of ATRA-sensitive gastric-cancer patients. The immune-regulatory responses activated by ATRA suggest that the retinoid and immune-checkpoint inhibitors constitute rational combinations for the management of gastric-cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-023-02869-w.

Original Western blots page 14-28 SUPPLEMENTARY METHODS

Short-term tissue slice cultures
The short term tissue slice cultures were prepared and used as already described (Centritto F, Paroni G, Bolis M, Garattini SK, Kurosaki M, Barzago MM, et al.Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression.EMBO Mol Med. 2015;7:950-72).Briefly, tissue slices (thickness, 200 μm) deriving from surgical specimens of 13 gastric cancer patients who underwent a diagnostic Tru-cut procedure were obtained within 24 hours from the resection.Tissue slices were challenged with vehicle (DMSO) or ATRA (1.0 μM) for 48 hours in a 1:1 mixture of DMEM/F12 medium containing EGF (20 ng/ml), FGF (20ng/ml), insulin (5µg/ml), glucose (0.3%) in the presence Antibiotic-Antimycotic solution (GIBCO).At the end of the treatment, samples were fixed, paraffin-included, and dissected into 5-μm slices, which were subjected to immuno-histochemical staining with an antibody targeting the Ki67 proliferation-associated marker.The percentage of Ki67-positive tumor cells in the various samples was assessed in a quantitative manner by automatic image analysis.Scoring of Ki67 was blinded as to treatment.Each value represents the mean ± SE of at least five separate fields for each experimental sample.S3 Characteristics of the gastric-cancer patients used for the studies involving tissue-slice cultures The table summarizes the clinical characteristics of the 13 patients considered.

Table S4 RNA-sequencing data
The table contains the processed RNA-seq data obtained with our panel of gastric cancer cell-lines exposed to vehicle and ATRA.

Table S5 Effects of ATRA on the expression of the RNAs derived from endogenous retroviruses
The table contains the levels of endogenous retroviral RNAs determined by RNA-seq data obtained from the indicated cell lines exposed to vehicle and ATRA.

Table S1 Characteristics and source of the gastric-cancer cell-lines
The cell-lines characterized by a G-INT and a G-DIFF phenotype are marked in blue and red, respectively.All the cell-lines used throughout the study were free from mycoplasma contamination.

Figure S4 HALLMARK pathway analysis of the RNA-seq results obtained following treatment of the indicated gastric cell lines with ATRA
Exponentially growing cultures of the indicated cell lines were exposed to ATRA (1.0 µM) for 48 hours.At the end of the treatment cells were subjected to RNA-seq analysis (Supplementary Table S3).

Figure S5 KEGG pathway analysis of the RNA-seq results obtained following treatment of the indicated gastric cell lines with ATRA
Exponentially growing cultures of the indicated cell lines were exposed to ATRA (1.0 µM) for 48 hours.At the end of the treatment cells were subjected to RNA-seq analysis (Supplementary Table S3).The data obtained were subjected to pathway analysis using the KEGG metabolic data set.The numbers shown indicate the Score values obtained.The FDR (False-Discovery-Rate) values are indicated in parenthesis.
When the FDR values are <0.1, they are considered to be statistically significant and they are marked in red with an asterisk.When the statistically significant pathways are up-regulated they are contained in a pinkbox.
By contrast, the down-regulated pathways are contained in light blue box.The most relevant up-regulated (red) and down-regulated (blue) pathways are contained in a yellow box.

Figure S1
Figure S1 Growth curves of the gastric cancer cell-lines exposed to increasing concentrations of ATRAThe indicated and exponentially growing cell-lines were exposed to vehicle or 5 increasing concentrations of ATRA (10 -9 to 10 -5 M) for 6 days.At the end of the treatment, cell-lines were subjected to MTS assays to determine their growth.Each value represents the Mean + SE of 10 independent cultures with the exception of HuG1-N, OCUM-1, SNU-1, SNU-5 and SNU-16 cells where each value is the Mean + SE of 4 independent cultures.The Area-Under-the-Curve (AUC) value, which was used for the calculation of the ATRA-scores, is indicated for each growth curve.

Figure S2
Figure S2 Ki67 immune-histochemistry in tissue-slice cultures of representative primary gastriccancers exposed to ATRA Tissue slices deriving from surgical specimens of 13 separate patients (P1-P13) were challenged with vehicle (DMSO) or ATRA (1.0 μM) for 48 hours.Tumor slices were fixed, paraffin embedded, cut into 5 µm slices and stained for the Ki67 protein using a specific antibody.The case characterized by a G-INT phenotype is marked in blue, the case characterized by a G-DIFF phenotype is marked in red.The figure illustrates examples of the immuno-histochemical data obtained in two representative cases: (i) Patient 1 (P1), G-INT case; (ii) Patient 2 (P2), G-DIFF case.

Figure S3
Figure S3 Effects of ATRA on the body weight of SCID mice transplanted with LMSU and NCI-N87 cells Ten Nude mice/experimental group were xeno-transplanted with human LMSU (A) and NCI-N87 (B) gastric-cancer cell-lines.Subsequently, mice were treated with vehicle (DMSO) or ATRA (15mg/kg) intra-peritoneally at the indicated time points (arrows).The total body weight of each animal was determined and the results are shown as the Mean+SD of the values.
The data obtained were subjected to pathway analysis using the HALLMARK data set.The numbers shown indicate the Score values obtained.The FDR (False-Discovery-Rate) values are indicated in parenthesis.When the FDR values are <0.1, they are considered to be statistically significant and they are marked in red with an asterisk.When the statistically significant pathways are up-regulated they are contained in a pink box.By contrast, the down-regulated pathways are contained in light blue box.The most relevant up-(red) or down-regulated (blue) pathways are contained in a yellow box.

Figure S6
Figure S6 Number of genes modulated by ATRA in retinoid-sensitive G-INT and G-DIFF gastric cancer cell-lines The G-INT/retinoid-sensitive GSU, KATO-III and IM95 cell-lines as well as the G-DIFF/retinoidsensitive HGC-27, LMSU, GCIY and RERF-GC-1B cell-lines were exposed to vehicle (DMSO) or ATRA (1.0 µM) for 48 hours.At the end of the treatment, cells were subjected to RNA-seq analysis.Left: The panel illustrates the number of genes selectively up-regulated (red) or down-regulated (blue) in each G-INT cell-line (squares) and commonly up-regulated (red) or down-regulated (blue) in the 3 cell-lines (circle).Right: The panel illustrates the number of genes selectively up-regulated (red) or down-regulated (blue) in each G-DIFF cell-line (squares) and commonly up-regulated (red) or down-regulated (blue) in the 4 cell-lines (circle).

Figure S7
Figure S7 Effects of ATRA on IRF1 protein levels and cell-growth in the retinoid-sensitive LMSU cell-line LMSU cells were transfected with two IRF1-targeting (si-IRF1a/si-IRF1b) and a control siRNA (si-CTRL).Twenty-four hours later, cells were treated with vehicle (DMSO) or ATRA (1µM) for 48 hours.Upper: Western-blot analysis using anti-IRF1 and anti-tubulin antibodies: the lanes marked as "no-siRNA" indicate the parental LMSU cells.The values shown underneath the IRF1 Western blots were obtained following densitometric analysis (Dens) of the IRF1 and Tubulin (Tub) bands and represent the IRF1/Tub ratio, as indicated.Lower: Cell-growth of the transfected LMSU cells (sulforhodamine-assay): the results are expressed as the Mean+SD values of 3 replicate cultures, all the values are normalized for vehicle-treated cells (100%).The p-values (two-tailed Student's ttest) of the comparisons between ATRA-treated and vehicle-treated cells are shown above each red column.The p-values are marked in red if they indicate statistical significance.The figure shows the data obtained in one of the three independent experiments performed, which provided identical results.

Figure S8
Figure S8 Effects of ATRA on IRF1 protein expression in retinoid resistant gastric cancer cells and IRF1 over-expression in AGS cells(A) The G-INT, OCUM-1, HuG1-N and AGS cell-lines as well as the G-DIFF, MKN-74 cell-line, which are characterized by a low level of sensitivity to the anti-proliferative effects of ATRA, were exposed to vehicle (DMSO) or ATRA (1.0 µM) for 48 hours.At the end of the treatment, cells were subjected to Western blot analysis with specific anti-IRF1and anti-tubulin (Tub) antibodies.The levels of tubulin in each lane of the gel are shown as a loading control.(B) Retinoid resistant AGS cells were transfected with a commercially available plasmid (myc-DDK-tagged human IRF1; Origene) allowing the expression of a tagged and biologically active form of the human IRF1 transcription factor (tag-IRF1) or the corresponding void vector (Vector).IRF1 expressing cells were selected in the presence of G418 (0.4 mg/ml) for 10 days.The Western blot shows the expression levels of the endogenous IRF1 protein and the over-expressed tag-IRF1 counterpart.(C) The entire Vector and tag-IRF1 cell populations were plated at the same cell density (10 4 cells/ml) in a 12-wells plate (triplicate cultures).Cells were grown in RPMI medium supplemented with charcoal treated Fetal Bovine Serum (FBS) in the absence of G418 for 2 days.Subsequently cells were exposed to vehicle (DMSO) or 1.0 µM for 6 days.The number of viable cells was determined with the use of a Beckman Coulter Counter (Vi-CELL BLU v1.4.2).Each value is the Mean+SD of 6 independent cultures.The p-value of the ATRA vs. vehicle comparison is shown above each red column.The comparisons between the results obtained in vehicle treated Vector and tag-IRF1 cells did not provide statistically significant results (p-value = 0.818).Similarly, the comparisons between the results obtained in ATRA treated Vector and tag-IRF1 cells did not provide statistically significant results (p-value = 0.443), as shown in the panel.

Table S2
Structure of the double stranded DNAs used for the construction of the shRNA plasmid constructsThe sequences of the sense and antisense strands of the oligonucleotides targeting the IRF1 and DHRS3 genes as well as the scramble control oligonucleotides (CTRL1 and CTRL2) are illustrated.
The nucleotides marked in red constitute the EcoRI and BamH1 sites used for the insertion of the double-stranded oligonucleotides into the pGreenPuro plasmid.The nucleotides marked in black represent the sequences corresponding to different targeted regions of the IRF1 (shIRF1a = exon 4, nucleotides 4-22; shIRF1b = exon 6, nucleotides 18-36) and DHRS3 genes (shDHRS3a = exon 3, nucleotides 54-72; shDHRS3b = exon 6, nucleotides 54-72).In shCTRL1 and shCTRL2 the nucleotides marked in black are scrambled and non-targeting oligonucleotides.In each oligonucleotide the black sequences are separated by an L12 loop marked in green.